The Ah receptor (AhR), a basic helix-loop-helix transcription factor, mediates the toxicity of 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD) and related xenobiotics. There is also compelling evidence that this protein normally functions in the regulation of cellular proliferation and differentiation processes. An understanding of AhR function, its regulation, and the mechanisms, by which it modulates gene expression, will significantly advance the ability to evaluate both the health risks from environmental levels of dioxin-like chemicals, and possible relationship of AhR-regulated processes to other disease states. There is substantial evidence that phosphorylation/ dephosphorylation processes regulate the functional activity of the AhR and that the AhR is a phosphoprotein. However, there are as yet no published data available to indicate specific phosphorylated sites on the AhR or their functional significance. It is hypothesized that direct phosphorylation of the AhR regulates its function as a transcription factor, and that these processes play a major role in tissue-, species-, and temporal-specific responses to the dioxin-like chemicals. The goal of these studies is to identify phosphorylated amino acid residues and to define their functional significance for the regulation of AhR transcriptional activity. Preliminary data indicates that there are at least four charged forms of the AhR. Using specific inhibitors and mutations known to affect AhR signaling and processing, studies will determine the relative presence of these forms at various phases of the signaling process. These data will suggest a function of these AhR forms and the possible pathways for the change in charge distribution. Additional experiments using MALDI-TOF mass spectrometry are designed to identify specific charged residues in the AhR. Finally, mutations of the AhR will be performed at known sites of phosphorylation and the functional significance of these mutations in terms of ligand binding, DNA binding, transactivation function, and AhR processing will be determined. Overall, data from these investigations will indicate the precise amino acid residues that are phosphorylated and their functional significance. ? ? ?
